THE FIRST STEP-BY-STEP PICTURE OF HOW DNA OPENS UP TO INTERACT WITH AN ENZYME.

Feb. 28, 1953: Two young men walk into a dingy pub in Cambridge, England called
the Eagle. To a lunchtime crowd they announce that they’ve discovered the
secret of life. They have.

The two young men were Watson and Crick. Fifty years later, their success at
deciphering the structure of DNA stands as the founding event of molecular
biology. The elegant spirals of this structure and the phrase that denotes it,
“the double helix,” have become ingrained in our culture. School
lessons teach us that the rungs of the DNA spiral staircase are bonded pairs of
chemical bases — A and T, C and G — letters that shape the destiny
of all known forms of life.

As this new science has progressed, we’ve learned much about how the
sequence of bases exerts its mighty influence, and we know that DNA
doesn’t act alone. Enzymes are the deus ex machina of DNA drama, coming
into the scene and inciting change. Enzymes interact with the bases to
facilitate cell division and protein-making, and as a first step in these
processes the base pairs must fold out from their sheltered space inside the
double helix, a structural shift called base flipping.

Alex MacKerell, Niu Huang, University of Maryland

“The information in DNA is hidden,” says University of
Maryland biophysicist Alex
MacKerell, “and for DNA to perform its biological function,
DNA has to open up so the information can be accessed. Base flipping
is a simple structural change that may be the first step in
replication and transcription of DNA and is essential for other
processes in which enzymes interact with the bases.”

Laboratory studies have shown the structure of flipped-out DNA, but
laboratory work tells virtually nothing of exactly what happens to
initiate the shift and what intermediate states occur along the way.
With the availability of LeMieux, Pittsburgh Supercomputing
Center’s terascale system, MacKerell and his research team
tackled these questions with an extensive series of simulations. Their
results — reported in the Proceedings of the National Academy of
Sciences (Jan. 7, 2003) — provide the first atom-by-atom,
step-by-step picture of enzyme-facilitated DNA base flipping.

Which Groove?

Click Image to Enlarge

This top-down view represents the unflipped DNA helix (right)
compared to the flipped state (left), with the target base
for methylation, cytosine (atoms in red), fully turned out.
The "orphaned" guanine (green atoms) remains within the
helix. Arrows indicate the major groove versus minor groove
pathway.

Although DNA base flipping happens in all organisms from plants to
people, researchers first confirmed it in bacteria. Laboratory studies
have shown that an enzyme called methyltransferase attaches to
cytosine, the C of A,T,C and G, and chemically changes it, by adding a
methyl group (CH3-). This relatively simple chemical
change, called methylation, is thought to be widespread in DNA
interactions. “We’re starting to understand,” says
MacKerell, “that chemical modifications of certain bases are
involved in the regulation of the expression and transcription of
DNA.”

The base has to flip out for methylation to occur, and the flipped-out DNA
structure has been identified in laboratory work. Still, what was known before
MacKerell’s work was a bare outline of the process, like seeing the opening
scene of a romantic movie and falling asleep until the noisy wedding at the
end.

A central unanswered question had to do with how much the enzyme is involved in
the base flipping. Does the enzyme help the base to flip out, or does it bind
after it’s already flipped? Experiments gave no clear answer. “How do we
understand,” asks MacKerell, “going from the normal duplex DNA shape to the
flipped out shape?”

Another question had to do with DNA’s grooves. Because of the way paired bases
stack up, an intact double helix of DNA has a groove on each side, one smaller
than the other, aptly named the minor groove, and a larger one called the major
groove. Through which of these grooves do the bases turn as they flip outward?
Structural evidence suggested the minor groove, but some experimental evidence
suggested the major groove.

“THE COMPUTER IS INVALUABLE, BECAUSE IT ALLOWS US TO LOOK AT
EVENTS, WHICH IN EXPERIMENTAL TIME FRAMES YOU CAN’T SEE. IN THE
COMPUTER WE CAN SEE WHAT HAPPENS.”

“This is where the computer is invaluable,” says MacKerell, “because it allows
us to systematically change the structure and look at events, which in
experimental time frames happen so fast that you can’t see them. In the
computer, using our mathematical models, we can see what happens.”

May the Force Field Be With You

To produce a comprehensive base-flipping picture, MacKerell turned to a
computational approach called molecular dynamics. In essence, MD treats a
molecule as a dynamic structure of atoms interacting with each other and with
nearby atoms. The computer tracks how each atom in the molecule moves by
calculating the forces between it and every other atom at successive slices of
time.

Over a period of years, MacKerell has helped to extend MD, first developed for
proteins, to become a powerful tool for DNA. Much of his work has focused on
“empirical force fields” — a way to express the
quantum-mechanical energies between atoms as empirical constants. Deriving
these empirical force fields, which approximate the probabilities of quantum
theory, is the art and science of MD. It has the important benefit of making it
possible to do MD simulations, which have proven ability to reveal the
atomic-level details of biomolecular processes.

“To get these parameters to treat the chemical system accurately,”
says MacKerell, “is a continual process in which we optimize the
empirical force field to reproduce experimental data. We also use quantum
mechanical data as part of the target data. The empirical force fields have
become more sophisticated and more accurate with time.”

Click Image to Enlarge

What Happens When the Enzyme Arrives
MacKerell’s simulations show that the methyltransferase
binding site — the part of the enzyme that interacts
with the DNA — has a dramatic impact on the free energy
of base flipping. When the enzyme (blue rods and coils) pairs
with the DNA helix, amino acids within the enzyme’s
binding-site loop (dark blue) interact directly with the
cytosine (red) and guanine (green). The first frame (top)
shows a serine (purple) competing for the hydrogen bonds
(dotted lines) that bind the two bases. The second frame
(above) shows the early flipped state, stabilized by the
serine and a glycine (gold).

To arrive at his full-story picture of base flipping, MacKerell broke the
process down into chunks he called “simulation windows.” Each
window is a scene from the full scenario. All possible configurations of the
DNA, from closed-to-flipped-to-closed, are represented as a circle, like a
clock face, which is sub-divided into 72 five-degree arcs. Within each of these
windows, MacKerell calculated relative free-energies, key information that
tells what shape the molecule prefers, since it tends to assume the shape that
requires the least expenditure of energy.

Using eight LeMieux processors for each free-energy window, MacKerell simulated
four different configurations of a 12-base DNA sequence: An unflipped helix in
a water solution (17,700 atoms), a flipped helix with methyltransferase in two
different positions, and a flipped helix with methyltransferase and a third
molecule, called a cofactor. For each five-degree window, he simulated 160
picoseconds (a trillionth of a second) of movement — with a snapshot of
the action every two femtoseconds, 80,000 time slices per window.

With fourteen months of computing time, 80,000 single-processor hours, and much
careful analysis, MacKerell and his colleagues had answers where before there
was only mystery. The enzyme initiates flipping, and the base flips through the
major groove pathway. “The presence of the enzyme destabilizes the
DNA,” says MacKerell, “and then the base interacts further with the
enzyme, until the enzyme-cofactor complex stabilizes the fully flipped
state.”

These findings, MacKerell believes, suggest a process by which DNA and the
enzyme are in cross talk with each other, like a molecular pas de deux. The
enzyme, arms spread, approaches to begin binding, and the DNA in turn starts to
open, which draws the enzyme closer, until it stabilizes the DNA in the flipped
state.

Overall, it’s a result that highlights the power of computational methods
to uncover the details of DNA-enzyme interactions, a field of study
that’s still new. “Everyone has known for a long time,” says
MacKerell, “that DNA has to change its shape to perform its function.
We’ve been able to show for the first time how an enzyme actually
facilitates the conformational change. And we’ve been able to see the
atomic details of how it does that.”

The Pittsburgh Supercomputing Center is a
joint effort of Carnegie Mellon University and the
University of Pittsburgh together with the Westinghouse
Electric Company. It was established in 1986 and is
supported by several federal agencies, the Commonwealth of
Pennsylvania and private industry.